The molecular pathogenesis of fully virulent, wild-type Y. pestis in relevant animal models has been relatively neglected because of the scarcity of secure BSL-3 facilities and trained personnel certified to work with this Class A select agent. The threat of bioterrorism and the emergence of multiply-antibiotic resistant strains of Y. pestis increases the urgency for a more detailed understanding of the host-pathogen relationship at the molecular level that may lead to the design of improved medical countermeasures and diagnostics. Y. pestis is one of the most invasive and virulent of bacterial pathogens. Bubonic plague pathogenesis is noted by an initial stealth phase, during which the bacteria multiply and spread to secondary lymphoid tissue without stimulating a strong innate immune response, followed soon thereafter by an aggression phase characterized by rapid systemic spread, hyperinflammation, and fatal septic shock. We have established mouse and rat models of bubonic plague that incorporate flea-to-rodent transmission to investigate the role of specific Y. pestis virulence factors and to characterize the host response to naturally acquired infection. We have characterized the kinetics, microbiology, and histopathology of bubonic plague in rats following intradermal injection of Y. pestis; and used this model to characterize the gene expression profile of Yersinia pestis in the infected lymph node during bubonic plague using whole-genome microarray technology. Our previous work has shown that three important Y. pestis virulence factors, Ail (a Y. pestis outer surface protein), the Type III secretion system (T3SS) encoded on the Yersinia virulence plasmid, and the plasminogen activator (Pla) encoded on the 9.5-kb Y. pestis-specific plasmid all act to limit the polymorphonuclear leukocyte response to bubonic plague infection in vivo (polymorphonuclear leukocytes, also referred to as PMNs or neutrophils, are phagocytic cells that are an important innate defense against infection). Thus, we now have several lines of evidence that the PMN response correlates with successful outcome to infection, and this aspect of host-pathogen interaction has become a focus of our lab. To facilitate our studies involving in vitro assays of Y. pestis-neutrophil interactions we have established a system in the lab for the generation of immortalized murine neutrophil progenitor cells based on retroviral transduction of a Hoxb8-estrogen receptor construct in to bone marrow cells described by Wang et al. (ref: Nat Methods. 2006 Apr;3(4):287-93). We have successfully used this method to generate large numbers of murine neutrophils and macrophages suitable for a variety of in vitro assays. We are currently working to characterize the response of murine neutrophils to Y. pestis and compare the results to those obtained in experiments using human neutrophils. We are also using murine neutrophils and macrophages to identify novel factors important for Y. pestis intracellular survival in vitro. During the past year, we continued using intravital microscopy to image the early host response to Y. pestis, focusing on bacterium-host phagocyte interactions in the skin-draining lymph nodes. Additionally, we have initiated a formal collaboration with Ron Germains group at NIAID to use the histocytometry and Ce3d tissue clearing techniques developed in his lab to further characterize the host response to Y. pestis in the lymph node. We will use these techniques to determine the fate of bacteria that arrive early after infection. We are expanding upon our earlier studies, which focused mainly on neutrophil responses to Y. pestis, to characterize monocyte, macrophage, and dendritic cell responses and interactions with Y. pestis in the lymph node as well. Both virulent and highly attenuated strains of Y. pestis can disseminate from the skin to the draining lymph node. The virulent strains replicate and can often eventually escape and spread systemically, whereas attenuated strains are contained and eventually killed. The containment mechanisms are not completely understood. We will evaluate the cellular response in the lymph node to virulent and attenuated strains and determine how pre-existing immunity to Y. pestis affects these responses. We will also determine how the cellular responses develop and change over the course of infection, especially during and after the prolonged infections we observed after flea-borne transmission. We continue to collaborate with Jason Cysters group at UCSF to determine the role of lymph node subcapsular sinus macrophages in the pathogenesis of bubonic plague. Dr. Cyster provided CD169-DTR transgenic mice that allow for depletion of subcapsular sinus macrophages using diphtheria toxin treatment. Preliminary results showed a significant decrease in the number of Y. pestis in the lymph nodes of subcapsular sinus macrophage-depleted mice compared to controls. However, we have struggled to replicate these results in a slightly different system using local injection of clodronate-loaded liposomes to deplete subcapsular sinus macrophages in the lymph node. Work in the coming year will focus on improving consistency of our mouse model and using it to gain a better understanding of the fate of Y. pestis immediately after dissemination to the lymph node. Specifically, we will determine whether or not subcapsular sinus macrophages provide a protective niche for Y. pestis in this tissue. Additionally, the histocytometry and tissue clearing methods we are using in collaboration with Ron Germains group will be very useful for examining Y. pestis- subcapsular sinus macrophage interactions in vivo. During FY2019 we finished a study that used an in vivo imaging system (IVIS) in conjunction with bioluminescent Y. pestis strains to monitor the incidence and dissemination patterns of infection in mice challenged by flea bite. Fleas were used within the first week after infection to evaluate what is known as early-phase transmission, and again 1 to 2 weeks after infection to evaluate a second, distinct transmission mechanism that is dependent on Y. pestis biofilm formation in the flea foregut. Results showed that flea-borne bubonic plague can follow an acute course, with dissemination from the flea bite site to the draining lymph node and systemic spread within a few days; or a prolonged course in which the bacteria multiply extensively in the intradermal flea bite site over several days, before either resolving or finally disseminating to cause systemic disease. This clinical picture differs from that seen following intradermal inoculation by needle, which invariably leads to acute disease. The chronic skin infection seen following flea bite challenge provides a new model to study immune responses in the dermis. During the last year we also initiated a project to evaluate the role of the Y. pestis F1 capsule in promoting mammal-to-flea transmission. In order to be transmitted, Y. pestis must infect the small blood capillary vessels in the superficial layer of the skin, which are the source of blood for a feeding flea. The internal diameter of these capillaries is only 4-6 micrometers. The objective of this study is to test the hypothesis that one function of the Y. pestis capsule is to prevent the formation of bacterial aggregates that would be inaccessible to a feeding flea because they are too large to readily enter into and flow through the small capillaries. According to this hypothesis, although the capsule is not required for virulence per se, it is important to complete the transmission cycle.